GSM7088804: PGCLCs, THC 100nM, biol rep 3; Mus musculus; RNA-Seq
Total RNA was extracted from ESCs and EpiLCs pellets using the AllPrep DNA/RNA Micro Kit (Qiagen), according to the manufacturer's protocol. For PGCLCs, d5 embryonic bodies were harvested and cells were dissociated using TrypLE™ Select followed by resuspension in fluorescence-activated cell sorting (FACS) buffer (1×Dulbecco's phosphate buffered saline [DPBS], 1% BSA, 1 mM EDTA, 25 mM HEPES) and cell suspension were passed through a cell strainer (70μm). Cells were sorted on a BD Biosciences FACSAria III (UCLA BSCRC Flow Cytometry Core). Practically, cell populations of interest, being double-positive (Blimp1::mVenus+ and Stella::ECFP+) were sorted and collected in microtubes containing GK15 medium. Total RNA was extracted from double-positive PGCLCs using the AllPrep DNA/RNA Micro Kit (Qiagen). Libraries were prepared with the KAPA mRNA HyperPrep Kit (BioMek) or with the RNA library prep kit (ABClonal) following the manufacturers' protocols. Briefly, poly(A) RNA were selected, fragmented and double-stranded cDNA synthesized using a mixture of random and oligo(dT) priming, followed by end repair to generate blunt ends, adaptor ligation, strand selection, and polymerase chain reaction (PCR) amplification to produce the final library.